Project Summary Many bacterial pathogens secrete exotoxins to modify the host-pathogen interactions in a manner that benefits the bacteria. There are several examples of successful prophylaxis and treatment by targeting bacterial toxins as evident by decades-long history of vaccines for tetanus, diphtheria, and pertussis. Staphylococcus aureus (SA) is a major human pathogen responsible for several hundred thousand of hospitalizations and over 12,000 death in the US every year. SA produces a plethora of toxins including pore-forming toxins (PFTs) that play a key role in pathogenesis and immune evasion. While neutralization of these toxins by monoclonal antibodies (mAbs) is expected to reduce clinical disease, it is unlikely to uproot invasive disease, as suggested by recent failure of a PFT mAb cocktail to prevent SA ventilator associated pneumonia (Arsanis clinical trial). We have devised a novel approach to target neutralizing anti-toxin mAbs specifically to the site of infection enabling instant toxin neutralization and mediating opsonophagocytic killing (OPK) at the same time. The approach exploits the cell wall targeting domains (CWT) of a phage-derived bacteriolysin which binds with species-specificity and high affinity to cell wall components of specific bacteria. The CWT is fused to specific anti-toxin mAbs to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). We have successfully applied this to B. anthracis and C. difficile under NIAID support. In this proposal we aim to develop ISTAbs for S. aureus using the isolated CWT of lysostaphin along with potent PFT neutralizing mAbs. ISTAbs are expected to accumulate where they are needed most, i.e. at the site of infection; they capture and sequester the toxins, thus immediately neutralizing the immune suppressive effects of the toxins and preventing their release into circulation. Bacterium-ISTAb-toxin complex is then cleared by phagocytes. Concurrent toxin neutralization and bacterial clearance is a unique advantage of the ISTAb technology over mere antitoxin treatment. In this proposal, we will use a set of broadly neutralizing S. aureus PFT mAbs to generate ISTAbs with lysostaphin CWT. However, because IgG binding to protein A on the surface of S. aureus interferes with opsonophagocytic killing and may even cause immunopathology, we will create and test mutant ISTAbs that do not bind protein A. In Aim 1, these ISTAbs will be generated and characterized for biophysical and functional properties, including neutralization and OPK activity, to select a short list for in vivo efficacy studies. In Aim 2, we will evaluate the efficacy of ISTAbs in different mouse models of S. aureus infection including pneumonia, sepsis and surgical wound infection. Upon completion of the Phase I SBIR we anticipate a Phase II project to expand the efficacy studies to invasive infection models in rabbits including sepsis, prosthetic joint infection, ventilator associated pneumonia, and endocarditis models as well as IND-enabling studies.